Preparation of toxic ricin



Oct. 23, 1962 H. L. CRAIG ETAL 3,060,165

PREPARATION OF TOXIC RICIN Filed July 3, 1952 LSlurry with waterI pH of3.8 sou using 5% u so,

LPrecipitate with N 80 pH of 7 using l2% Na,CO,

l Wash filter cake with |6.7% M0 80 I Single extraction Wash solutionExtract with wateri pH of 3.8#O.l using H 80 iii Precipitate with Na IpHof 7 using i2% Na ()0 [E'E l Wash filter cake with l6.7% No.80 I

Wash solution Grind cake to 40 mesh Slurry with CCI.

| Settle and skim oft Ricin i Settled M0 reuse I N VEN TORS Harry L.Craig 0. h. A/derks Alsop/l H. 00min Sally h. Die/re 0h affe L. Karel BY4 14 W ATTORNEY Dry and grind States. ate

This invention relates to the method of preparing toxic llClIl.

Ricin is a protoplasmic poison prepared from castor beans after theextraction of castor oil therefrom. It is most effective as a poisonwhen injected intravenously or inhaled, the latter requiring extremecomminution and small particle size to be effective. It is believed thatthe toxic action is catalytic rather than stoichiometric which probablyaccounts for the high toxicity of the agent.

Because of its relative instability, ricin must be handled with extremecare. In neutral aqueous solution it is stable only up to 60-75 C., andin solid form up to 100110 0, although for short exposures, temperaturesup to 130 may be tolerated. It is sensitive to acids, alkalis andhalogen and may also be inactivated by mechanical working such asgrinding or pulverizing. These factors are of great importance indeveloping a satisfactory method for preparing the material.

Although ricin has been prepared in crystalline condition in thelaboratory in small quantities, it becomes necessary, for purposes oftoxicological warfare, to prepare relatively large quantities in a highstate of purity. This necessitates that as much as possible of thenon-toxic material present be removed in the process.

In preparing the protein material, the castor beans are first ground andpressed to remove most of the oil. The pressed cake still retains about15% oil and this may be removed by means of solvents which will extractan additional 150 pounds of oil per ton of beans and reduce the oilretained in the cake to a little over 1%. In the event that theexpressing step is supplemented by solvent extraction, it is importantto prevent detoxification of the protein during the solvent removalstep. If residual solvent is removed from the ground beans by blowingwith steam, considerable detoxification results. Blowing with nitrogenefiectively prevents detoxification but is expensive when carried out ona large scale.

After the oil has been removed, the pressed cake or pomace is extractedby agitating with water at a pH of 3.8101 at 25 C. which removessubstantially all of the toxic protein. The extraction process isoperative within a pH range of about 3 to 4.5 although the preferredrange is about 3.5 to 4., The optimum operating point is a pH of3.8-1.1, as indicated above. A careful pH control is essential in orderthat as much non-toxic protein as possible may be eliminated and alsothat the filtration rate may be held at a satisfactory value. Either HClor H 80 may be used to get the desired pH for the extraction water, butH 50 is preferred due to its lower corrosion rate and ease of handlingin concentrated form. The acid should be used in reasonably dilute formto prevent undue local concentrations during its addition. A 5%concentration is satisfactory.

Following the extraction, the slurry is filtered using either aconventional recessed plate filter or a continuous string dischargevacuum filter. With the latter about 7% of filter aid, based on mealweight, was found necessary for satisfactory filtration.

The filtrate from the water extraction step, which contains the ricin,was treated with a 16.7% solution of Na SO to precipitate the protein.This solution is com- "ice posed of 20 pounds of salt in 100 pounds ofwater and the amount used was such that the salt content equalled 20% ofthe filtrate weight. This amount and concentration of salt solution wasabout optimum considering the factors of cost and toxin recovery.Somewhat higher concentrations and larger amounts of solution can beused, however.

The precipitation process is not limited to the use of Na SO since asaturated solution of NaCl can be used successfully, but Na SO solutiongives better nitrogen fractionation, more rapid precipitation, and canbe operated under wider pH limits. It is desirable to raise the pH toabout 7-8 before precipitation as this gives better ecovery and greaternon-toxic nitrogen removal. The pH was raised to this value by usingNaOH or Na CO the latter being preferred. The base used was quite dilutein order to prevent detoxification due to high local concentrations inthe solution. A 5% solution of NaOH was used, whereas with Na CO a 12%solution was preferred. In general, this higher pH during precipitationgave a greater non-toxic nitrogen fractionation and at the same timemaintained the toxin loss at less than 2%.

After precipitation, the slurry was filtered using from 1 to 4% filteraid, based on slurry weight, for satisfactory filtration; the amount offilter aid needed being dependent on the type of press used. Washing thefilter cake with Na SO solution removed additional non-toxic nitrogenwhich is desirable. In this washing step a 16.7% solution of Na SO wasagain used. This washing step removed an additional 15% of non-toxicnitrogen from the cake.

After filtration the filter cake, which contains the ricin incombination with the Na SO may be dried and slurried with CCL, toseparate the ricin by flotation. Separation of the ricin after a singleprecipitation and washing step is possible, but it is preferred to carrythe process through an additional extraction and precipitation step.This is accomplished by slurrying the filter cake in three times itsweight of water and the pH of the slurry is again brought to 3.8:.1 bymeans of 5% H The slurry is filtered and a second precipitation isbrought about by adding Na SO solution. Although pH control here is notwholly essential it is advantageous to bring the pH to approximateneutrality by adding 12% Na CO A precipitation time of 45 minutes wasnecessary to obtain complete removal of the toxin. In filtering out theprecipitate, no filter aid was used and the filter cake was washed withNa SO solution on the filter whereby an additional amount of nontoxicnitrogen was removed from the cake. This washing was effective only thefirst time and repeated washings had little effect in removing furthernon-toxic nitrogen.

The ricin-Na SO precipitate was dried at about 50 to 60 C. on a hot airtray dryer. The dried product was ground to pass a 40 mesh screen andagitated with 5 times its weight of CCl.;, which served the separate thericin from the Na SO by flotation. After settling, the ricin was skimmedoff the top. This reduced the Na SO content of the mixture from aprevious 40 to 50% down to 15 to 18%. About 1 to 2% of nitrogen remainedin the Na SO salt which could then be used for subsequentprecipitations.

The final precipitation produced a particle size of 1-2 mu. On dryingthe wet cake, however, the ricin cemented together forming largerparticles. These could not be broken down to their original size byordinary grinding methods and since a very fine particle size wasnecessary in order that the product might be used as a toxic weapon, itwas thought desirable to seek some method to prevent the agglomerationor cementing process that took place on drying.

To attempt to affect this result, physical conditions prevailing underthe precipitation process were changed.

This included changing the temperature of precipitation and the rate ofagitation. Other changes included precipitation with ony partialsaturation of Na SO and the use of wetting and seeding agents. None ofthese expedients produced any significant improvement in particle size.

Ordinary dry ball and hammer milling of the dried ricin producedconsiderable detoxifiiation perhaps due to the generation of excessheat. The use of CCl slurry plus the use of low temperature and lowmoisture content of the ricin reduced detoxification during ballmilling.

Spray drying proved to be an even better method of securing a reasonablysmall particle size. Best results Were achieved by using a solutionhaving about 20% solids, an inlet temperature of 150 C. and an atomizingair pressure of 150 to 180 psi. The particle size secured was 6 to 8 mu.

The best means of securing a small particle size was by air grinding.This was carried out in an apparatus having a chamber with conical topand bottom. The material to be ground has been fed into this chamber andis withdrawn from the bottom and forced back into the center of thechamber tangentially through a venturi. Compressed air of about 100 psi.was fed to the venturi to provide the grinding force. The fines aredrawn off the top and the large particles settle to the bottom to berecirculated and reground. This process produced particles having a massmedian diameter of 2.5 to 3.5 mu.

Numerous variations are possible in the several steps of the processcommencing with the water extraction and precipitation which may be asingle or multiple step. Although a single extraction step can be used,as indicated before, some process modifications are necessary for itssuccessful operation on a plant scale. Double extraction proved to bequite efiicien-t but additional steps beyond the second extraction stepwere not found necessary.

The drawing is self-descriptive and shows the various steps of theprocess described.

We claim:

1. In a method of preparing toxic ricin from castor beans comprisingslurrying an expressed castor bean cake with water to remove the watersoluble ricin and precipitating the ricin from the filtrate, the furthersteps which include slurrying the precipitate with CCl and separatingthe ricin by flotation.

2. A process in accordance with claim 1 in which the precipitate isdried prior to slurrying.

References Cited in the file of this patent J. Gen. Physiol, vol. 32(1948), pages 25-31.

1.IN A METHOD OF PREPARING TOXIC RICIN FROM CASTOR BEANS COMPRISINGSLURRYING AN EXPRESSED CASTOR BEAN CAKE WITH WATER TO REMOVE THE WATERSOLUBLE RICIN AND PRECIPI-TATING THE RICIN FROM THE FILTRATE, THEFURTHER STEPS WHICH INCLUDE SLURRYING THE PRECIPITATE WITH CC14 ANDSEPARATING THE RICIN BY FLOTATION.